The present invention relates to methods and compositions for the reduction of xenotransplantation rejection. Specifically, the present invention relates, first, to transgenic cells, tissues, organs and animals containing transgenic nucleic acid molecules that direct the expression of gene products, including, but not limited to enzymes, capable of modifying, either directly or indirectly, cell surface carbohydrate epitopes such that the carbohydrate epitopes are no longer recognized by natural human antibodies or by the human cell-mediated immune response, thereby reducing the human immune system response elicited by the presence of such carbohydrate epitopes. In a preferred embodiment, the transgenic cells, tissues, organs and animals express nucleic acid molecules encoding functional recombinant xcex1-Galactosidase A (xcex1GalA) enzyme which modifies the carbohydrate epitope Galxcex1(1,3)Gal. In a more preferred H embodiment, the transgenic cells, tissues, organs and animals expressing the functional recombinant xcex1GalA are transgenic pig cells, organs, tissues and/or animals. Second, the present invention relates to methods for xenotransplantation comprising introducing the transgenic cells, tissues and/or organs into human recipients so that a lower level of hyperacute rejection (HAR) is observed in the human recipients relative to the level of HAR observed in human recipients having received non-transgenic cells, tissues and/or organs. The invention is demonstrated by way of the Examples presented in Sections 6-11, below, which, for example, describe the expression of functional recombinant xcex1GalA in transgenic cells and the corresponding dramatic reduction of cell surface Galxcex1(1,3)Gal carbohydrate such expression causes (Sections 7 and 10), further demonstrate that transgenic cells expressing functional recombinant xcex1GalA elicit a significantly reduced level of complement-mediated cytoxicity (Section 9), and still further demonstrate that transgenic xcex1-galA dramatically reduces the level of Galxcex1(1,3)Gal in vivo.
The severe shortage of human organs available for transplantation purposes has led to a great interest in the use of animal-to-human organ transplants, termed xe2x80x9cxenotransplantsxe2x80x9d. Extensive studies now exist regarding such xenotransplantations. See, e.g., Sandrin et al. (Sandrin, M. S. et al., 1994, Transplant. Rev. 8:134), which discusses studies involving the use of pig organs for xenotransplantation to humans.
The body""s first reaction to a foreign tissue, known as hyperacute rejection (HAR), is a rapid and severe one, and represents one of the largest obstacles to the success of xenotransplantation techniques. HAR is for the most part, is mediated by antibodies and complement, there being natural human antibodies, predominantly IgG and IgM subclasses, which react with numerous molecules on xenotransplant cells, particularly endothelial cells, in vascularized transplants (Cooper, D. K. C. et al., 1994, Immunol. Rev. 141:31; Sandrin, M. S. and McKenzie, I. F. C., 1994, Immunol. Rev. 141:169). It is now generally accepted that all or most of the HAR reaction is due to the presence of human antibodies directed against the carbohydrate epitope Galxcex1(1,3)Gal. This has been shown by absorption studies, particularly with Gal+ transfected cells, and by the fact that Galxcex1(1,3)Gal carbohydrates can block the reaction in vitro and in vivo (Sandrin, M. S. et al., 1994, Xenotransplantation 1:81).
Attempts to eradicate HAR have included removal or neutralization of complement in various procedures, using Cobra venom factor or transgenic animals expressing human complement regulatory molecules (e.g. CD46, CD55 and CD59). Other approaches have included the logistically difficult removal of antibody (Oriol, R. et al., 1993, Transplantation 56:433), and attempts to alter the antigen itself. With respect to this latter approach, the gene encoding the pig xcex1(1,3)galactosyltransferase, which is necessary for the production of the GAlxcex1 (1,3) Gal carbohydrate, has been isolated with the aim of performing gene knockout studies by homologous recombination. Unfortunately, such procedures cannot be done in the pig. See Sandrin et al. (Sandrin, M. S. et al., 1994, Transplant. Rev. 8:134) for a review of other approaches to prevent the expression of the Galxcex1(1,3)Gal, which include the use of anti-sense constructs but these have met with variable, in general disappointing results.
Another approach which has been attempted makes use of the enzyme xcex1-Galactosidase A which cleaves a terminal xcex1-linked galactosyl residue (Oriol, R. et al., 1993, Transplantation 56:433). Treatment of red blood cells, lymphocytes and endothelial cells with xcex1-Galactosidase A inhibits their reaction with human serum and Cairns et al have demonstrated a similar phenomenon in vivo. A meeting report has reported that perfusion of tissue prior to transplantation with the bacterial xcex1-Galactosidase A enzyme delayed the onset of HAR (Cairns, T. et al., 1994, Transplant. Proc. 26:1279). These enzyme treatment approaches, however, are difficult. For example, the enzymes are expensive and perfusion with an enzyme prior to transplant would be difficult, in reality, to accomplish, particularly in a manner which would ensure total eradication of the epitope.
In summary, therefore, while xenotransplantation represents a potential solution to the severe shortage of human donor organs which currently exists, the problem of hyperacute rejection continues to be a major obstacle to the successful use of xenotransplantation.
The present invention relates to methods and compositions for the reduction of xenotransplantation rejection.
Specifically, the present invention relates, first, to transgenic cells, tissues, organs and animals containing transgenic nucleic acid molecules representing functional carbohydrate epitope-modifying genes which direct the expression of gene products that, either directly or indirectly, bring about modification of cell surface carbohydrate epitopes, including, but not limited to the Galxcex1(1,3)Gal cell surface carbohydrate epitope, in a manner which reduces the human immune system response elicited by the resulting modified epitope relative to that response elicited by the unmodified Galxcex1(1,3)Gal epitope. Such gene products can include, but are not limited to, carbohydrate epitope-modifying enzymes capable of modifying cell surface carbohydrate epitopes such that the carbohydrate epitopes are no longer recognized by either natural human antibodies or the human cell-mediated immune system, thereby reducing the human immune system response elicited by the presence of such carbohydrate epitopes.
In a preferred embodiment of the invention, the transgenic cells, tissues, organs and animals express transgenic nucleic acid molecules encoding functional recombinant xcex1-Galactosidase A (xcex1GalA) enzyme which modifies the carbohydrate epitope Galxcex1(1,3)Gal by cleaving the terminal xcex1-linked galactose from the carbohydrate epitope prior to its transfer to the cell surface on different molecules, thus producing cells which are phenotypically Galxcex1(1,3)Gal minus. In a more preferred embodiment, the transgenic cells, tissues, organs and animals expressing the functional recombinant xcex1GalA are transgenic pig cells, organs, tissues and/or animals. In yet another preferred embodiment of the invention, xcex1GalA and H transferase genes are co-expressed in the transgenic cells, tissues, organs and animals of the invention.
Second, the present invention relates to methods for xenotransplantation comprising introducing the transgenic cells, tissues and/or organs into human recipients so that a lower level of hyperacute rejection (HAR) is observed in the human recipients relative to the level of HAR observed in human recipients having received non-transgenic cells, tissues and/or organs, thereby reducing the level of xenotransplantation rejection.
The invention is demonstrated by way of the Examples presented in Sections 6-11, below, which describe the expression of functional recombinant xcex1GalA in transgenic cells and the corresponding dramatic reduction of cell surface Galxcex1(1,3)Gal carbohydrate such expression causes (Sections 7 and 10), further demonstrate that transgenic cells expressing functional recombinant xcex1GalA elicit a significantly reduced level of complement-mediated cytoxicity (Section 9), and still further demonstrate that transgenic xcex1-galA dramatically reduces the level of Galxcex1(1,3)Gal in vivo.
The transgenic cells, tissues, organs and animals of the invention can serve a variety of functions. For example, the transgenic cells, tissues and organs of the invention can be used as xenotransplants for introduction into human recipients. The transgenic animals of the invention can be used as sources for xenotransplant material to be introduced into human recipients or, alternatively, as sources for the production of transgenic cell lines. Alternatively, specific transgenic cells of the invention, namely bone marrow cells, may be used to produce red blood cells exhibiting an altered ABO phenotype, that is, can convert blood group B erythrocytes into erythrocytes of universal donor group O.
The term xe2x80x9cfunctional carbohydrate epitope-modifying genexe2x80x9d, as used to herein, refers to a nucleic acid sequence which encodes and directs the expression of a gene product that, either directly or indirectly, brings about modification of a cell surface carbohydrate epitope, including, but not limited to, the Galxcex1(1,3)Gal cell surface carbohydrate epitope, in a manner which reduces the human immune system response elicited by the resulting modified epitope relative to that response elicited by the unmodified Galxcex1(1,3)Gal epitope.
The term xe2x80x9cfunctional carbohydrate epitope-modifying enzymexe2x80x9d, as used to herein, refers to an enzyme, encoded by a functional carbohydrate epitope-modifying gene, which modifies a cell surface carbohydrate epitope, including, but not limited to the Galxcex1(1,3)Gal cell surface carbohydrate epitope, in a manner which reduces the human immune system response elicited by the resulting modified epitope relative to that response elicited by the unmodified Galxcex1(1,3)Gal epitope.
The term xe2x80x9cfunctional xcex1GalAxe2x80x9d or xe2x80x9cfunctional recombinant xcex1GalAxe2x80x9d, as used to herein, refers to an xcex1GalA enzyme which modifies the cell surface carbohydrate epitope Galxcex1(1,3)Gal in a manner which reduces the human immune system response elicited by the resulting modified epitope relative to that response elicited by the unmodified Galxcex1(1,3)Gal epitope.